A number of intestinal tract-derived peptide hormones are known. Among them, VIP (vasoactive intestinal polypeptide) is known to have favorable pharmacologic activities, for example vasodilating, hypotensive and bronchodilating activities. VIP shows little variation in peptide sequence among animal species, for example, human VIP and porcine VIP are nearly identical in sequence. VIP is found not only in the intestinal tract but also in many other organs. Recently, it has become recognized that VIP may serve as a neurotransmitter.
However, because of a Met (L-methionine) residue in the molecule, naturally occurring human VIP is unstable to oxidation. Furthermore, VIP is susceptible to enzymolysis which results in inactivation. Therefore, investigations have been made on finding various derivatives of VIP that exhibit greater stability.
The present inventors made intensive investigations in an attempt to obtain peptides having naturally occurring human VIP-like activities by modifying the peptide sequence of VIP without impairing the activities of VIP and introducing one or more amino acids which would not preclude production of the peptide by recombinant DNA technology. As a result, it was found that novel peptides of the sequence:
H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-V al-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Pro-R, wherein R is Glu-Ala-OH, Glu-OH or OH (SEQ ID NOs.: 1 to 3, respectively) these peptides are equal or superior to human VIP from the viewpoint of pharmacologic activity and, in addition, are stable in vivo.
Meanwhile, at present, physiologically active peptides generally are produced by extraction from natural sources, by organic synthetic means, or by utilizing recombinant DNA or genetic engineering techniques.
However, large-quantity production of physiologically active peptides with a molecular weight of 2,000 to 10,000 by extraction from animal organs or by organic chemical synthesis is not cost effective. In producing such peptides by genetic engineering techniques, when they are expressed as such in prokaryotic cells, the peptides readily decompose resulting in low recoveries. Therefore, recombinant peptides have had to be recovered by an inefficient process comprising first producing fusion proteins between the desired peptide and .beta.-galactosidase, for instance, followed by cleavage thereof and purification of the resulting desired peptides (for example, Simoncsits et al, Eur. J. Biochem., vol 178, pp 343-350 (1988) and JP-A-01-296996 (The term "JP-A" as used herein means an "unexamined published Japanese patent application")).
The present inventors made intensive investigations on the production method of gene manipulation and found that large-quantity production of L-methionine-free peptides with a molecular weight of 2,000 to 10,000 becomes possible when a fused protein composed of a fragment of the galK protein, which fragment covers 200 to 300 amino acid residues from the N terminus of said protein, and any of the above-mentioned peptides having a molecular weight of 2,000 to 10,000 as coupled to said fragment at the C terminus thereof via a L-methionine residue, can be expressed in prokaryotic cells using the trp promoter. The fused protein is purified, treated with cyanogen bromide and said peptide, 2,000 to 10,000 in molecular weight, is purified from the resulting peptide mixture.